Angiogenesis is the process of generating new capillary blood vessels. During angiogenesis, neovascularization is tightly regulated and activation thereof occurs in embryogenic development, tissue remodeling, wound healing, and periodic cycles of corpus luteum development (Folkman and Cotran, Int. Rev. Exp. Pathol., 16, 207–248, 1976).
During the process of angiogenesis, capillary blood vessel endothelial cells start to proliferate from an existing vasculature. The endothelial cells grow very slowly as compared with other types of cells in a body. The proliferation of these cells is induced by pro-angiogenic cytokines, inflammation mediators, and activated proteolytic enzymes.
Failure to regulate angiogenesis leads to the development of several clinical syndromes or conditions. Pathological angiogenesis is involved in various diseases such as cancer in metastatic phase, arthritis, psoriasis, and retinopathy.
Not only reorganization of the blood vessel by migration, proliferation and differentiation of endothelial cells, but also degradation of an extracellular matrix is required for angiogenesis. One of the major events for inducing angiogenesis is a breakdown of the extracellular matrix before the formation of the capillary blood vessels. One of the most important enzymes which are involved in the matrix degradation is matrix metalloproteinase (MMP), a family of over 20 proteins. MMPs are endopeptidases, which degrade or proteolyze various components of the extracellular matrix such as collagen, proteoglycan, and gelatin.
An MMP's activity is modulated by an endogenous substance called Tissue Inhibitors of Metalloproteinases (TIMP) (Liotta and Stetler-Stevenson, Semin. Cancer. Biol. 1(2), 99–106, 1990; Liotta et al., Cell, 64(2), 327–336, 1991). The proteins in the TIMP family are classified as tumor suppressor proteins and four proteins have been identified as members of this family.
TIMP2, one of the four identified proteins in TIMP family, is able to bind to pro- and active form of MMP-2. Since TIMP2 inhibits all the activated forms of MMPs, TIMP2 acts as a key inhibitor molecule in angiogenesis and cancer metastasis. For example, tumor cell growth and metastasis were inhibited by a gene therapy with TIMP2 in experimental animals (Hajitou et al., Cancer Res., 61, 3450–3457, 2001; Li et al., Human Gene Ther. 12, 515–526, 2001; Sacco et al., Gene Ther., 8, 67–70, 2001). However, studies with TIMP2 were very limited due to a very limited amount of the protein existing in a biological system. Therefore, it is indispensable to develop a recombinant technique for overexpressing the TIMP-2 protein in vitro.
Although E. coli is a preferred host in recombinant DNA technology for producing large quantities of heterologous proteins economically, certain foreign proteins expressed in large quantities from E. coli are precipitated as inclusion bodies. Recovery of a biologically active protein from these inclusion bodies has presented critical problems and the recovered proteins are often biologically inactive because they are folded into a three-dimensional conformation different from that of native protein. Since TIMP2 has 6-disulfide linkages, it is very complicated to refold denatured TIMP2 into its correct, biologically active conformation.
As a eukaryote, yeast is a suitable host organism for a high-level production of secreted soluble cytosolic proteins of human origin. Indeed, many kinds of pharmaceutically important proteins have been expressed in yeast. Yeast is able to splice out introns and transport proteins through secretory pathways as higher eukaryotes do. Especially, Saccharomyces cerevisiae, the molecular and cellular biology of which has been intensively studied, has been exploited as a host for heterologous protein production since essential elements for gene expression such as strong and regulable promoters, vectors, and genetic markers are well developed (Romanos et al., Yeast, 8, 423–488, 1992). Moreover, its use in food fermentation for thousands of years proved that S. cerevisiae causes no harm to human beings and the processes for the production of therapeutic proteins using yeast acquired GRAS (generally recognized as safe) status. Altogether, these features make S. cerevisiae one of the most suitable organisms for heterologous gene expression.
Despite many advantages of yeast expression systems, a number of proteins are neither expressed in a large quantity nor secreted efficiently in yeast for unknown reasons. When human TIMP2 is expressed in yeast, for example in S. cerevisiae, the expression level is extremely low.
Human serum albumin (HSA) consisting of 585 amino acids is the most abundant protein in plasma, representing about 60% of total plasma proteins. A major function of serum albumin is to maintain a natural osmotic pressure of plasma and to transport sparingly soluble substances throughout the body. Serum albumin also functions as a carrier of endogenous and exogenous molecules, and for many years it has been thought to be devoid of any enzymatic function. However, recently, it has been found that it acts as dihydrotestosterone enolase and phospholipid cysteine peroxidase (Drmanovic et al., Anticancer Res. 19(5B), 4113–4124, 1999; Hurst et al., Biochem J., 338(Pt3), 723–728, 1999). Despite these findings, exogenously administered modified serum albumins, for example recombinant therapeutic proteins fused to serum albumin, are not likely to contribute significantly to the total albumin pool because of the relative abundance of albumin in plasma. Furthermore, human serum albumin is a very stable protein displaying an in vivo half-life of 19 days in the adult human (Sterling, K., J. Clin. Invest., 30, 1228, 1957).